The overall goal of the research is to determine the mechanism of replacement of sulfur with selenium in a tRNA molecule. Previous work in this group showed that a selenium atom replaces sulfur in modified E. coli tRNAs, which have a 5-methylamino-methyl-2-thiouridine base in the wobble position of the anticodon of tRNA(Glu) or tRNA(Lys). It is known that the selenium donor is selenophosphate, which can be made by selenophosphate synthetase from ATP and selenide, and that a new enzyme called 2-selenouridine tRNA synthase catalyzes the replacement of sulfur with selenium in the appropriate tRNA molecules. One important study in this reaction will be to obtain direct evidence of the mechanisms by using radiolabeled 35S tRNA as the enzyme substrate. The determination of the labeled product should be a strong indicator of the reaction mechanism. In this case, we are looking initially for (35S)thiophosphate, which is a reasonable product to expect, since no ATP is required for the reaction and since selenophosphate is the selenium donor. The steps required in this work include: the growth and isolation of selenophosphate sythetase from E. coli; the growth and isolation of 2- selenouridine tRNA synthase; the growth, isolation, and quantitative study of the modified tRNAs in E. coli; and finally the reaction with 35S-labeled tRNA and the determination of the labeled product.